Homogeneous photoelectrochemical sensing platform and application thereof

Abstract

The application belongs to the technical field of biological detection, and relates to a homogeneous photoelectrochemical sensing platform and application thereof.The homogeneous photoelectrochemical sensing platform comprises a PyTTA-HOF / MXene modified working electrode, a magnetic signal transduction probe, a hairpin DNA, a primer RNA, a Cas12 enzyme and a buffer solution.The application combines the homogeneous amplification driven by the CRISPR / Cas12a system trans-cleavage with the excellent hydrogen peroxide PEC response of the PyTTA-HOF / MXene photoelectrode, and utilizes the gold nanoparticle loaded GOx to construct a "signal opening" PEC label, realizes the super-sensitive detection of human papilloma virus nucleic acid, avoids the complicated electrode modification step, and can be extended to other biomarkers by replacing the hairpin probe recognition domain, and has the advantages of high sensitivity, simple operation and universality.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a homogeneous photoelectrochemical sensing platform and its applications. Background Technology

[0002] Photoelectrochemical (PEC) technology combines the high sensitivity of photoexcitation with the low cost, low background interference, and ease of miniaturization of electrochemical detection, attracting widespread attention in the field of analytical sensing. Based on this, photoelectrochemical biosensing technology, combining the high specificity of biorecognition with the high sensitivity of photoelectric signals, has become one of the important research directions in modern analytical chemistry, showing broad potential in the high-precision quantitative analysis of various biomolecules such as DNA, proteins, enzymes, and cells. However, further development of this technology still faces two key challenges: first, the need to develop photoactive materials with efficient photogenerated carrier separation and transport capabilities to achieve efficient conversion between light capture and target recognition signals; second, the need to overcome the problems of high interfacial mass transfer resistance, low electron transfer efficiency, and decreased biorecognition dynamics due to steric hindrance caused by traditional bioprobe immobilization strategies on photoelectrode surfaces. Therefore, developing novel high-performance photoactive materials and constructing new homogeneous photoelectrochemical sensing strategies that do not require immobilization and directly respond to target analytes in solution is of great significance for promoting the development of this field.

[0003] In recent years, framework materials such as metal-organic frameworks (MOFs), covalent organic frameworks (COFs), and hydrogen-bonded organic frameworks (HOFs) have become ideal material platforms for constructing high-performance photoelectric sensors due to their high specific surface area, tunable pore structure, and ease of functionalization. Among them, HOFs are assembled entirely from organic units through non-covalent interactions such as hydrogen bonding, offering advantages such as strong structural designability, mild synthesis conditions, and environmental friendliness. In particular, hydrogen-bonded networks can promote charge delocalization within and between molecules, which is beneficial for improving the separation and migration efficiency of photogenerated electron-hole pairs, providing unique potential for their application in the field of photoelectric sensing. However, due to the relatively weak nature of hydrogen bonding interactions, traditional HOF materials often face problems such as insufficient stability and limited photoelectric performance, resulting in their research and application in the field of photoelectric sensing still being in its early stages. It is urgent to improve their stability and photoelectric performance through reasonable structural design to expand their application prospects in this direction. Summary of the Invention

[0004] The purpose of this invention is to provide a homogeneous photoelectrochemical sensing platform and its applications, thereby overcoming the shortcomings of existing technologies. By combining the CRISPR / Cas12a system with the superior PyTTA-HOF / MXene, a multifunctional and universal homogeneous photoelectrochemical sensing platform is developed, exhibiting significant flexibility and versatility. It is anticipated that by replacing the hairpin recognition domain, it can be directly extended to other nucleic acid biomarkers. This not only provides advanced photoactive materials and homogeneous sensing concepts for future photoelectrochemical biological detection but also offers valuable insights for developing highly sensitive, user-friendly diagnostic tools.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a homogeneous photoelectrochemical sensing platform, comprising a working electrode modified with MXene-functionalized PyTTA-HOF material (PyTTA-HOF / MXene), a magnetic signal transduction probe, hairpin DNA, primer RNA, Cas12a enzyme, and a buffer solution. The magnetic signal transduction probe consists of streptavidin magnetic beads and gold nanoparticles modified with glucose oxidase attached to their surface.

[0007] This invention deeply integrates the homogeneous exponential signal gain driven by the CRISPR / Cas12a system through reverse cleavage with the excellent hydrogen peroxide photoelectrochemical (PEC) response of the PyTTA-HOF / MXene photoelectrode. It utilizes CRISPR / Cas technology to amplify the detection signal and leverages the significantly enhanced performance of PyTTA-HOF material through MXene functionalization to construct a detection platform that combines high sensitivity, extremely low background interference, low-cost electrochemical readout, and ease of miniaturization. In particular, gold nanoparticles modified with glucose oxidase (GOx) act as "signal-on" PEC tags and bind with streptavidin magnetic beads to further enhance signal transduction efficiency, achieving a synergistic effect between homogeneous amplification and heterogeneous PEC response.

[0008] In some other embodiments, the streptavidin magnetic beads and glucose oxidase-modified gold nanoparticles are linked by a DNA linker; the 5' end of the DNA linker is modified with biotin, and the 3' end is modified with a thiol group.

[0009] This invention utilizes the high affinity binding of biotin at the 5' end of the DNA linker to streptavidin magnetic beads, while simultaneously constructing a "signal activation" tag through covalent coupling of the 3' end thiol group of the DNA linker to gold nanoparticles (AuNPs). This approach combines the rapid separation and enrichment advantages of streptavidin magnetic beads with the modular design flexibility of DNA linkers, ensuring both high specificity and low background interference in the detection system. Furthermore, it amplifies the detection signal through enzyme catalysis, significantly improving the sensitivity and ease of operation of biosensing.

[0010] In some other embodiments, the Cas12a enzyme has trans-cleavage activity against DNA linkers; The buffer solution is a Tris-HCl buffer solution with a pH of 7.4.

[0011] As an example, the hairpin DNA comprises H1 and H2, the nucleotide sequence of H1 being shown in SEQ ID NO:1; the nucleotide sequence of H2 being shown in SEQ ID NO:2. The sequence of the primer RNA is shown in SEQ ID NO:4; and the nucleotide sequence of the DNA linker is shown in SEQ ID NO:5.

[0012] In some other embodiments, the working electrode modified with MXene-functionalized PyTTA-HOF material is prepared by adding an aqueous solution of MXene-functionalized PyTTA-HOF material to the working electrode and drying it with infrared radiation. Specifically, the working electrode is indium tin oxide (ITO). Before adding the MXene-functionalized PyTTA-HOF material aqueous solution, the working electrode is first washed and dried. That is, it is ultrasonically washed with dilute hydrochloric acid, sodium hydroxide, acetone, methanol and ethanol solutions in sequence, and then vacuum dried to improve the stability of the bonding between the MXene-functionalized PyTTA-HOF material and the electrode.

[0013] The preparation method of the MXene-functionalized PyTTA-HOF material (PyTTA-HOF / MXene) is as follows: PyTTA monomer solution is added dropwise to MXene aqueous solution and stirred and mixed, then centrifuged, washed, and freeze-dried to obtain the material. The molar ratio of PyTTA to MXene is (3-4):1, preferably 3.6:1.

[0014] Specifically, the preparation method of PyTTA-HOF / MXene is as follows: MXene colloidal solution is dropped into deionized water and ultrasonically dispersed; PyTTA monomer solution is then dropped into the MXene aqueous solution while stirring, and stirring continues. PyTTA-HOF / MXene is collected by centrifugation. The PyTTA monomer solution is a DMF solution of PyTTA.

[0015] This invention enables in-situ self-assembly of PyTTA-HOF and MXene through simple drop-addition and stirring. This not only avoids the damage to the layered structure of MXene and the porous framework of PyTTA-HOF caused by harsh conditions such as high temperature and strong acid and alkali, but also utilizes the surface functional groups of MXene and π-π stacking to guide the uniform growth of PyTTA-HOF between layers / on the surface, effectively inhibiting MXene stacking and forming a tightly structured heterogeneous interface. At the same time, the process is simplified and energy-efficient, and the composition and structure of the composite material can be flexibly controlled by adjusting the raw material ratio and reaction parameters. Ultimately, a functionally synergistic reinforcing material with both the high conductivity of MXene and the high specific surface area of ​​PyTTA-HOF is obtained, and it is suitable for large-scale preparation.

[0016] In some other embodiments, the magnetic signal transduction probe is prepared by sequentially adding a gold nanoparticle solution, a DNA linker, and streptavidin magnetic beads to a glucose oxidase solution, shaking to mix, and then magnetically separating, washing, and dispersing in a buffer solution for storage.

[0017] By using DNA linkers (with biotin modified at the 5' end and thiol modified at the 3' end) to bridge streptavidin magnetic beads and glucose oxidase-modified gold nanoparticles, a magnetic signal transduction probe combining magnetic separation and enrichment functions with enzyme catalytic signal amplification capabilities was successfully constructed. This not only ensured stable loading of GOx and gold nanoparticles to enhance signal intensity, but also significantly reduced non-specific adsorption through the rapid separation characteristics of streptavidin magnetic beads. This enabled the low-cost, high-efficiency preparation and highly sensitive detection application of the magnetic signal transduction probe.

[0018] Secondly, the present invention provides the application of the homogeneous photoelectrochemical sensing platform of the first aspect in the detection of biological target molecules.

[0019] This homogeneous photoelectrochemical sensing platform decouples the trans-cleavage activity of the CRISPR / Cas12a system from the photoelectrochemical signal output. It can be adapted to different nucleic acid targets simply by replacing the recognition domain of the hairpin probe, demonstrating a universal detection capability for a variety of nucleic acid biomarkers.

[0020] In some other embodiments, the detection method of biological target molecules is as follows: the sample to be tested is mixed and incubated with hairpin DNA to prepare a mixed solution; a Cas12a enzyme-primer RNA complex is added to the mixed solution, and after mixing and incubation, a magnetic signal transduction probe is added, and after mixing and incubation, the mixture is separated by magnetic force to obtain a supernatant containing GOx-modified gold nanoparticles; a buffer solution is added to the supernatant to prepare an electrolyte, and a three-electrode system consisting of a working electrode modified with MXene-functionalized PyTTA-HOF material is used for photoelectric testing under ultraviolet irradiation, and the content of biological target molecules in the sample to be tested is calculated according to the standard curve.

[0021] In some other embodiments, the biological target molecule is human papillomavirus nucleic acid, which is one or more of HPV-16, HPV-18, HPV-31, HPV-51, and HPV-58, and the sample to be tested includes serum.

[0022] Specifically, this PEC sensing platform is used for HPV-16 detection at 1.0 × 10⁻⁶. -14 Up to 1.0×10 -8 Ultrasensitive quantitative detection of HPV-16 within the M concentration range, with a detection limit (LOD) as low as 4.8 × 10⁻⁶. -15 M (Signal-to-noise ratio S / N=3).

[0023] In some other embodiments, the incubation time for adding the Cas12a enzyme-primer RNA complex is 20-100 min, and the incubation time for adding the magnetic signal transduction probe is 20-70 min. Preferably, the incubation time for adding the Cas12a enzyme-primer RNA complex is 60 min, and the incubation time for adding the magnetic signal transduction probe is 60 min.

[0024] In some other embodiments, Ag / AgCl is used as the reference electrode and Pt as the counter electrode in the three-electrode system, and the wavelength of ultraviolet irradiation is 360-370 nm. Specifically, the wavelength of ultraviolet irradiation is 365 nm.

[0025] The beneficial effects of this invention are: (1) The sensor prepared a magnetic signal transduction probe (MSP) with both magnetic separation function and enzyme catalytic signal amplification capability through biotin-avidin bridging, and innovatively linked hybridization chain reaction (HCR) with the trans-cleavage function of CRISPR / Cas12a system: the target triggers HCR to form double-stranded DNA to activate Cas12a enzyme, and its non-specific cleavage releases GOx-AuNPs. GOx loaded in GOx-AuNPs catalyzes glucose to produce H2O2 as a photoelectrochemical (PEC) detection signal. This strategy achieves efficient coupling of homogeneous reaction and heterogeneous detection through magnetic separation technology, which avoids cumbersome electrode modification and significantly improves detection sensitivity by using a dual amplification mechanism. Moreover, through modular DNA sequence design, it can be flexibly adapted to different targets, showing high versatility and clinical application potential.

[0026] (2) This integrated scheme can achieve the detection of human papillomavirus (HPV) DNA at 1.0 × 10⁻⁶ m². -14 Up to 1.0×10 -8 Ultrasensitive quantitative detection within the M concentration range, with a detection limit (LOD) as low as 4.8 × 10⁻⁶. -15M (Signal-to-noise ratio S / N=3). A solution-phase-based PEC biosensing platform without immobilization was successfully established. Benefiting from the combination of CRISPR / Cas12a system-mediated homogeneous biorecognition and PyTTA-HOF / MXene photoluminescence within a single container, this strategy effectively bypasses cumbersome electrode modifications while significantly improving detection performance in a satisfactory manner. More importantly, this sensing strategy exhibits significant flexibility and versatility—it is expected to be directly extended to other nucleic acid biomarkers by replacing the hairpin recognition domain. Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1 This is a schematic diagram of a homogeneous photoelectrochemical sensing strategy; Figure 2 The images show the structural characterization of PyTTA-HOF and PyTTA-HOF / MXene, where A is a scanning electron microscope (SEM) image of PyTTA-HOF, B is a scanning electron microscope (SEM) image of PyTTA-HOF / MXene, C is an XRD pattern of PyTTA-HOF and PyTTA-HOF / MXene, D is an elemental distribution map of PyTTA-HOF / MXene, E is an XPS image of MXene, PyTTA-HOF and PyTTA-HOF / MXene, and F is a Fourier transform infrared (FTIR) spectrum of MXene, PyTTA-HOF and PyTTA-HOF / MXene. Figure 3 The image shows the high-resolution XPS spectrum of PyTTA-HOF / MXene, where A represents C 1s, B represents O 1s, C represents F 1s, D represents Ti 2p, and E represents N 1s. Figure 4 The figures show the optical performance characterization of PyTTA-HOF and PyTTA-HOF / MXene, where A is the UV-vis DRS diagram of PyTTA-HOF and PyTTA-HOF / MXene, B is the Tauc diagram of PyTTA-HOF, C is the Tauc diagram of PyTTA-HOF / MXene, D is the VB-XPS diagram of PyTTA-HOF, E is the VB-XPS diagram of PyTTA-HOF / MXene, and F is the electronic band structure diagram of PyTTA-HOF and PyTTA-HOF / MXene. Figure 5The image shows the characterization of the magnetic signal transduction probe. In the image, A is the UV-Vis absorption spectrum of the precursor Au, precursor GOx, and the supernatant after MSP assembly (the inset shows the color change of the solution during MSP assembly); B is the elemental distribution of MSP; C is the Zeta potential of MB and MSP (the inset shows the magnetic properties of MSP); and D is a scanning electron microscope image of MSP. Figure 6 The diagram shows the optimization of experimental conditions, the effect of incubation time, and the error bar represents the standard deviation of three repeated measurements. Among them, A is the optimization diagram of the CRISPR / Cas12a system activation experimental conditions, and B is the optimization diagram of the magnetic signal transduction probe cutting experimental conditions. Figure 7 This is a feasibility test diagram for the photoelectric sensing platform. A shows a comparison of the photoelectric response of PyTTA-HOF and PyTTA-HOF / MXene; B shows the photoelectric response of PyTTA-HOF / MXene in the absence of hydrogen peroxide and 10... -7 M is the photoelectric response diagram under hydrogen peroxide conditions, C is the photoelectric response diagram of PyTTA-HOF / MXene under different concentrations of hydrogen peroxide (the inset shows the linear calibration curve of photocurrent intensity versus the logarithm of hydrogen peroxide concentration; the error bars represent the standard deviation of three parallel experiments), and D is a schematic diagram of the photoelectric response mechanism involving hydrogen peroxide. Figure 8 This is a quantitative detection graph of the target DNA, where A represents the PyTTA-HOF / MXene pair with no target and 10 -8 The graphs show the photoelectric response of target M, B, and C. respectively. B represents the photoelectric response of the photosensor to different concentrations of target DNA. C is a linear calibration curve of photocurrent intensity versus the logarithm of HPV-16 concentration. Error bars represent the standard deviation of three parallel experiments. D represents the specificity of the photosensor: a is blank, b is 1.0 × 10⁻⁶. -10 M HPV-18, c is 1.0×10 -10 M HPV-31, d is 1.0×10 -10 M HPV-51, e is 1.0×10 -10 M HPV-58, f is 1.0×10 -11 M HPV-16 and g are a mixture of af; E is the photoelectric sensor pair of 1.0×10 - 13 The graph shows the stability of the photoelectric response of the M HPV-16 sensor, and F represents the photoelectric sensor response to 1.0 × 10⁻⁶. -13 Long-term stability test diagram of the photoelectric response of M HPV-16. Detailed Implementation

[0029] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0030] The inventive concept of this invention is to construct a novel homogeneous photoelectrochemical (PEC) biosensing platform based on the synergistic effect of MXene-modified hydrogen-bonded organic frameworks (HOFs / MXene) and the CRISPR / Cas12a system, in order to solve the problems of insufficient structural stability of HOFs, low photogenerated carrier efficiency and limited homogeneous biorecognition signal transduction in the prior art.

[0031] Specifically, this invention utilizes the two-dimensional properties, metallic conductivity, and abundant surface end groups (=O, -OH, -F) of MXene to modify HOFs (such as PyTTA-HOF) through hydrogen bonding and π-π interactions. This significantly enhances the structural stability of HOFs to resist hydrolytic deformation and suppresses electron-hole recombination, thereby improving photogenerated carrier efficiency. Building upon this, the invention innovatively combines the PyTTA-HOF / MXene composite material with the CRISPR / Cas12a system. Utilizing the trigger chain of the Cas12a enzyme to induce trans-cleavage activity (which can indiscriminately cleave non-target single-stranded DNA), it transforms biorecognition (such as specific RNA / DNA detection) into a tunable photoelectric signal. This platform achieves efficient signal transduction and amplification through homogeneous reactions, possessing high sensitivity, specificity, and versatility, providing a novel sensing strategy for molecular diagnostics.

[0032] The operational principle of this strategy is as follows: Figure 1As shown, firstly, the MXene-functionalized PyTTA-HOF material PyTTA-HOF / MXene was easily synthesized via solvent-induced self-assembly, and its excellent photoelectric conversion performance was verified. A magnetic signal transduction probe (MSP) for signal transduction and output was designed by linking streptavidin magnetic beads (MB) with glucose oxidase-loaded gold nanoparticles (GOx-AuNPs) via a DNA linker (Bio-ssDNA-SH). This study selected human papillomavirus type 16 (HPV-16) DNA as a model analyte, as this high-risk oncogenic sequence biomarker is crucial for cervical and oropharyngeal cancer. Two rationally designed self-folding hairpin structures were used to mediate homogeneity, and trigger DNA was generated through exponential target recovery amplification, subsequently activating the CRISPR / Cas12a system's trans-cleavage activity against Bio-ssDNA-SH. Non-specific cleavage of DNA linkers releases GOx-AuNPs from the MSP surface into solution. The GOx loaded in these particles can catalyze the generation of photoelectrochemically active hydrogen peroxide from glucose, which can be rapidly quantified by a PyTTA-HOF / MXene photoelectrode. This paves the way for rapid one-step biosensors that combine the PyTTA-HOF / MXene photoelectrochemical platform with amplification technology driven by a CRISPR / Cas12a system.

[0033] Example 1 1. Experimental Section (1) Medicines and reagents: Indium tin oxide (ITO) coated glass was supplied by Zhuhai Kaiwo Optoelectronic Technology Co., Ltd. (Zhuhai, China); hydrochloric acid, acetone, sodium hydroxide, methanol (≥99.5%), anhydrous ethanol (≥99.5%), and N,N-dimethylformamide (DMF) were purchased from Yantai Yuandong Fine Chemical Co., Ltd. (Yantai, China); 1,3,6,8-tetra-(p-aminophenyl)pyrene (PyTTA) was purchased from Jinan Tudafei Trading Co., Ltd.; multilayer Ti3C2 colloidal aqueous solution (MXene) was purchased from Beijing Beike New Materials Technology Co., Ltd.; tetrachloroauric acid... Hydrogen peroxide (H2O2) and HAuCl4 were purchased from Aladdin (Shanghai); glucose oxidase (GOx) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) was purchased from Sigma-Aldrich; tris(hydroxymethyl)aminomethane was purchased from Tianjin Kemei Chemical Reagent Co., Ltd.; Cas12a enzyme was purchased from Guangzhou Megbio Biotechnology Co., Ltd.; streptavidin magnetic beads (MB) were purchased from Shanghai Titan Technology Co., Ltd.; all reagents were of analytical grade and could be used directly without further purification. HPLC-purified DNA was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). For example, the nucleotide sequences used are detailed in Table 1.

[0034] Table 1 DNA Sequence

[0035] (2) Instruments: Zahner Zennium Pro workstation (Zahner PP211, Zahner Ennium Pro, Germany), RigakuSmartLab X-ray diffractometer, Scanning electron microscope (SEM) (Thermo Fisher Scientific FIB-SEM GX4), Talos F 200 X (FEI, USA) transmission electron microscope (TEM), D / Max-2500 diffractometer K-Alpha spectrometer (Thermo Science Ltd, USA, XRD), Hitachi UV-Vis-NIR spectrophotometer (UH4150), Nicoleti S50 infrared spectrometer, Zetasizer Nano ZSP nanoparticle size potentiometer.

[0036] 2. Experimental Procedure (1) Preparation of PyTTA-HOF and PyTTA-HOF / MXene Specific synthesis procedure: Weigh 5 mg of PyTTA monomer and sonicate it into 0.5 mL of DMF. While stirring, add the PyTTA monomer solution dropwise into 10 mL of deionized water. Continue stirring for 1 hour, and collect the product PyTTA-HOF by centrifugation. Wash the resulting yellow product four times with deionized water and freeze-dry.

[0037] 100 μL of 5 mg / mL MXene colloidal solution was added dropwise to 10 mL of deionized water and ultrasonically dispersed for 30 minutes. PyTTA monomer solution was then added dropwise to the MXene aqueous solution while stirring. Stirring was continued for 1 hour, and PyTTA-HOF / MXene was collected by centrifugation. The resulting dark green product was washed four times with deionized water and then freeze-dried.

[0038] (2) Preparation of magnetic signal transduction probe Preparation of gold nanoparticles: 100 mL of 1 mM HAuCl4 solution was placed in a three-necked flask and heated to boiling with vigorous stirring. After boiling, 10 mL of 38.8 mM sodium citrate solution was quickly added, and stirring was continued for 20 minutes until the solution turned dark red. The solution was then cooled and stored at 4 °C for later use.

[0039] Preparation of the magnetic signal transduction probe: First, 100 μL of Bio-ssDNA-SH (1 μM) was heated at 95 °C for 5 minutes, cooled to 25 °C, and then mixed with 20 μL of TCEP (10 mM) in a shaker at room temperature for 1 hour. Simultaneously, 200 μL of 0.5 mg / mL GOx solution was injected into 200 μL of gold nanoparticle solution and shaken at room temperature for 1 hour. This solution was then mixed with the thiol-activated Bio-ssDNA-SH and shaken for another 12 hours. Next, 20 μL of 10 mg / mL streptavidin magnetic beads (MB) was added, and the mixture was shaken for another 2 hours. After magnetic separation, the probe was washed three times with 10 mM Tris-HCl (pH=7.4) buffer and redispersed in 200 μL of 10 mM Tris-HCl (pH=7.4) buffer to obtain the magnetic signal transduction probe, which was stored at 4 °C for later use.

[0040] (3) Preparation of photoelectrode Indium tin oxide (ITO) was sequentially ultrasonically washed with dilute hydrochloric acid, sodium hydroxide, acetone, methanol, and ethanol solutions for 10 min each, and then dried under vacuum at 60 °C for 1 hour. A PyTTA-HOF / MXene aqueous solution was ultrasonicated for five minutes to obtain a homogeneous colloidal solution. 10 μL of the colloidal solution was dropped onto an ITO electrode region with a circular area of ​​0.5 cm radius, dried under an infrared lamp for 20 min, and then stored for later use.

[0041] (4) H2O2 detection PyTTA-HOF / MXene / ITO was used as the working electrode, Ag / AgCl as the reference electrode, and Pt as the counter electrode. A 365 nm UV lamp was used as the light source, and 0.02 M Tris-HCl buffer (pH=7.4) was used as the electrolyte for photoelectric testing.

[0042] A certain amount of 30% H2O2 solution was placed in 0.02 M Tris-HCl (pH=7.4) solution to prepare a solution with a concentration of 10. -3 Solution M was serially diluted tenfold to a concentration of 10%. -7 10 -8 10 -9 10 -10 and 10 -11 The photoelectrode constructed was used to study the photoelectric response of the H2O2 solution of M to the above H2O2 solutions of different concentrations.

[0043] (5) Target DNA detection First, 4 μL of target DNA solutions of different concentrations were mixed with 8 μL of 1 μM H1 solution and 8 μL of 1 μM H2 solution and incubated at 37 °C for 2 hours. The Cas12a enzyme-primer RNA complex (100 nM) was prepared by incubating 10 μL of 200 nM Cas12a enzyme (2 μL of 10× buffer) with 8 μL of 200 nM primer RNA (RNase-free buffer) at 37 °C for 30 minutes.

[0044] 6 μL of H1-H2 mixed solution was mixed with 6 μL of Cas12a enzyme-primer RNA complex (100 nM) and incubated at 37 °C for 1 hour. Then, 100 μL of magnetic signal transduction probe was added to 12 μL of the mixed solution and incubated with shaking at 37 °C for 1 hour. After magnetic separation, the supernatant containing glucose oxidase-modified gold nanoparticles was obtained. The solution was prepared into 1 mL electrolyte by adding 0.02 M Tris-HCl (pH=7.4) buffer. Photoelectric detection was performed using a three-electrode system (PyTTA-HOF / MXene / ITO as the working electrode, Ag / AgCl as the reference electrode, and Pt as the counter electrode) and a 365 nm UV lamp as the light source.

[0045] 3. Results and Discussion (1) Crystal structure and morphology PyTTA-HOF and PyTTA-HOF / MXene were characterized using scanning electron microscopy (SEM). Figure 2 As shown in Figure A, the structure of PyTTA-HOF consists of micron-sized elongated strips. After in-situ synthesis of MXene, scanning electron microscopy (SEM) revealed that the prepared PyTTA-HOF / MXene material exhibits a large number of micron-sized elongated strips distributed on the surface of the MXene nanosheets. Figure 2 (B in the image). In addition, the element distribution map (...) Figure 2 Figure D) shows that PyTTA-HOF / MXene contains C, N, O, and Ti. Experimental results indicate that PyTTA-HOF and MXene successfully composite to form PyTTA-HOF / MXene. On one hand, the functional groups (-OH, -F, =O) on the MXene surface can form hydrogen bonds with the amino groups in the PyTTA-HOF backbone, thereby promoting the growth and loading of PyTTA-HOF on its surface. On the other hand, the conjugated aromatic ring structure in the PyTTA-HOF backbone may form π-π stacking interactions between PyTTA-HOF and MXene when close to the MXene sheets. However, the main forces involved in the composite process require further analysis based on material characterization.

[0046] The successful preparation of PyTTA-HOF / MXene was further verified by powder X-ray diffraction (XRD). Figure 2 As shown in Figure C, the powder diffraction peaks of PyTTA-HOF are consistent with the standard PyTTA-HOF card reported in the literature, confirming the successful synthesis of PyTTA-HOF material. For the PyTTA-HOF / MXene composite material, its diffraction peak at 2θ=6.35° differs slightly from the diffraction peak of PyTTA-HOF at 2θ=6.50°. Considering the characteristic diffraction peaks of MXene reported in the literature, the slight difference may be due to the overlap and shift between the diffraction peaks of the MXene (002) crystal plane and the diffraction peak of PyTTA-HOF at 2θ=6.50°. Furthermore, the sharp peak appearing at 2θ=26.98° in PyTTA-HOF / MXene may be attributed to rutile (PDF#21-1276), indicating that MXene underwent oxidation during the synthesis process. In summary, PyTTA-HOF / MXene exhibits almost all the characteristic peaks of PyTTA-HOF and MXene, and no additional peaks were observed in the spectrum of the composite, indicating that the two form a stable composite material.

[0047] The surface elemental composition and chemical structure of MXene, PyTTA-HOF, and PyTTA-HOF / MXene were analyzed using X-ray photoelectron spectroscopy (XPS). Figure 2 As shown in E, PyTTA-HOF / MXene clearly exhibits characteristic peaks corresponding to the elements C, O, N, F, and Ti.

[0048] The molecular structures, chemical bonds, and functional groups of MXene, PyTTA-HOF, and PyTTA-HOF / MXene were characterized by Fourier transform infrared spectroscopy (FI-IR). Figure 2 (F in the text). For MXene, it is at 3449 cm. -1 and 1632 cm -1 The absorption peaks at these locations correspond to the stretching vibration of hydrophilic OH molecules and the bending vibration of adsorbed water molecules, respectively. However, PyTTA-HOF retains the 3445 cm⁻¹ peak, which is characteristic of the NH stretching vibration. 1 and 3377 cm 1 The two signals at this location indicate the presence of a free -NH2 end group on the benzene ring. The center is at 2923 cm⁻¹. -1 The peak at 3213 cm⁻¹ can be attributed to the OH stretching vibration of NH₃···O. 1 and 3027cm 1 The peak at 833 cm⁻¹ can be attributed to the NH stretching vibration of NH···N, which together support the presence of hydrogen bonds in PyTTA-HOF. -1 The peak at 1616 cm⁻¹ is a characteristic peak of the para-disubstituted benzene ring, and because there are four such structural units in the PyTTA monomer molecule, this peak is extremely prominent. -1 The peak at 800 cm⁻¹ represents both the NH bending vibration peak of the amino group and the C=C stretching vibration peak of the aromatic ring. MXene at 800 cm⁻¹ -1 The following peaks correspond to Ti-C and Ti-O vibrations, confirming the presence of functionalized MXene sheets. For the PyTTA-HOF / MXene composite, the FT-IR spectrum reveals characteristics of both parent materials, indicating successful integration of PyTTA-HOF with MXene. Interestingly, compared to PyTTA-HOF, the characteristic infrared peaks representing hydrogen bonding are weakened, suggesting that the primary force in the composite of PyTTA-HOF and MXene may not be hydrogen bonding but rather π-π stacking interactions.

[0049] Figure 3 High-resolution XPS spectra of C 1s, O 1s, F 1s, Ti 2p, and N 1s of the PyTTA-HOF / MXene composite were presented. Compared with the original MXene nanosheets, the detection results of nitrogen (N) in PyTTA-HOF / MXene further validated the successful synthesis of the PyTTA-HOF / MXene composite. As shown in the high-resolution N 1s spectrum, PyTTA-HOF / MXene exhibited a characteristic peak corresponding to -NH2 at 399.73 eV and a peak corresponding to NH3 at 400.80 eV. + Characteristic peaks ( Figure 3 In addition, characteristic peaks corresponding to C-Ti, CC, CN, CO, and OC=O were observed in the high-resolution C 1s at 282.36 eV, 284.78 eV, 285.77 eV, 286.33 eV, and 290.43 eV. Figure 3 (A) Photoelectron signals of O 1s element were observed at 530.95 eV, 532.88 eV, 533.85 eV, and 534.83 eV, corresponding to Ti-O, C=O, CO, and O, respectively. -CO-C characteristic peak ( Figure 3 (B in the image). High-resolution F 1s shows that F atoms are mainly bonded to Ti surface groups, exhibiting a characteristic peak at 685.39 eV corresponding to Ti-F (…). Figure 3(C in the text). As shown in the high-resolution Ti 2p diagram, the peaks appearing near 465.48 eV and 462.11 eV confirm the presence of Ti 2p. 1 / 2 The presence of Ti 2P was confirmed near 456.96 eV and 455.72 eV. 3 / 2 The presence of [a specific substance] is shown at 455.72 eV, 456.96 eV, and 459.73 eV, corresponding to C-Ti-C, C-Ti-(O / F), and TiO, respectively. 2-x- F 2x Characteristic peaks ( Figure 3 (D in the text). For the Ti 2p region, there is a certain amount of TiO. 2–x –F 2x This bond may be due to MXene oxidation.

[0050] (2) Optical characteristics and optical performance To investigate the differences in photoelectric properties between PyTTA-HOF and PyTTA-HOF / MXene, the optical properties of the materials were first studied using solid-state UV-Vis diffuse reflectance (UV-Vis DRS). Solid-state UV-Vis diffuse reflectance testing revealed the light absorption range and capabilities of PyTTA-HOF and PyTTA-HOF / MXene. Figure 4 (A) In comparison, it was found that PyTTA-HOF / MXene has better light absorption capacity than PyTTA-HOF. According to Kubelk Based on Munk theory, further processing of solid-state UV-Vis diffuse reflectance experimental data yielded the bandgap widths of PyTTA-HOF and PyTTA-HOF / MXene. Figure 4 B and Figure 4 In the C section, compared to PyTTA-HOF, the band gap width of PyTTA-HOF / MXene is narrowed to a certain extent ( Figure 4 The F in the image is likely due to MXene doping. Furthermore, the VB-XPS spectra of PyTTA-HOF and PyTTA-HOF / MXene (…) Figure 4 D and Figure 4 The E) treatment was performed to obtain the valence band positions of the two materials. No significant difference was found between their valence band positions, indicating that MXene doping does not significantly affect the valence band position. In summary, MXene coupling reduces the band gap from 2.32 eV to 1.96 eV and decreases the maximum valence band value from 1.45 eV to 1.12 eV. This enhances light harvesting and photogenerated charge density, thus supporting the excellent photoelectrochemical (PEC) activity of the PyTTA-HOF / MXene biosensor interface.

[0051] (3) Characterization of magnetic signal transduction probe The successful construction of MSP was initially verified using ultraviolet-visible spectroscopy. For example... Figure 5 As shown in Figure A, the precursor solution of gold nanoparticles (AuNPs) and the glucose oxidase (GOx) solution exhibit characteristic absorption peaks at 520 nm and 280 nm, respectively. After assembly, both absorbance values ​​of the MSP supernatant decreased significantly, indicating that these components were effectively captured by MB. Figure 5 The color change shown in illustration A provides visual verification of this process. Zeta potential monitoring ( Figure 5 Figure B shows that the zeta potential of the modified MB changed from -9 mV to -20 mV, which is consistent with the enrichment of negatively charged DNA and gold nanoparticles (AuNPs) on the surface. Scanning electron microscopy (SEM) Figure 5 As shown in C), these distinct bright spots are uniformly distributed on the MSP surface, with an elemental distribution ( Figure 5 D) further verified the presence of Fe, P, and Au, confirming the integration of DNA with gold nanoparticles on the MB scaffold and confirming the successful formation of MSP. Furthermore, as... Figure 5 As shown in illustration B, the excellent magnetism of the magnetic signal transduction probe is verified by the visually apparent color difference.

[0052] (4) Optimization of experimental conditions In the photoelectrochemical detection process, the incubation time for activation of the CRISPR / Cas12a system and the incubation time for cutting the magnetic signal transduction probe were optimized. The error bar represents the standard deviation of three repeated measurements. Figure 6 As shown in Figure A, the photocurrent reaches its maximum at an incubation time of 60 minutes as the activation incubation time of the CRISPR / Cas12a system increases. Figure 6 As shown in Figure B, the optimal incubation time for non-specific cleavage of the magnetic signal transduction probe by the CRISPR / Cas12a system is 60 minutes. To obtain better analytical results, 60 minutes was consistently used as the incubation time for CRISPR / Cas12a system activation and magnetic signal transduction probe cleavage.

[0053] (5) Design of photoelectric sensing platform A homogeneous photoelectric sensing research strategy based on PyTTA-HOF / MXene composite material coupled with a CRISPR / Cas12a system, and the construction of a scheme for detecting HPV-16 are as follows: Figure 1As shown, a signal transduction probe with separation function was prepared by indirectly linking GOx and MB through biotin-avidin interaction, Au-S bonds, and Au-N bonds. In the presence of the target analyte, the target DNA and hairpin DNA open H1 through complementary base pairing, adding H2. The resulting double-stranded DNA H1-H2 forms a ternary complex with the primer RNA, thereby activating the CRISPR / Cas12a system. Simultaneously, the target DNA is released and continues to participate in the opening of H1, forming a signal amplification cycle. The activated CRISPR / Cas12a system non-specifically cleaves the magnetic signal transduction probe (MSP). Since H2O2 and target DNA satisfy a quantitative relationship, after magnetic separation, the H2O2 produced by glucose catalyzed by the GOx modified on the released gold nanoparticles is used as a detection signal for photoelectrochemical (PEC) measurement. This sensor employs a homogeneous interface sensing strategy using magnetic separation technology, avoiding excessive electrode modification. By using hybridization chain reaction (HCR) and the CRISPR / Cas12a system to non-specifically cleave single-stranded DNA, it achieves target signal amplification and detection, significantly improving the sensor's detection sensitivity. Furthermore, by changing the DNA sequence, it can detect different targets, expanding the sensor's application range.

[0054] (6) Feasibility test of photoelectric sensing platform To verify the significantly enhanced PEC properties of PyTTA-HOF doped with MXene, photoelectric tests were conducted using a 0.02 M Tris-HCl buffer solution as the electrolyte, with PyTTA-HOF / ITO and PyTTA-HOF / MXene / ITO as the working electrodes, respectively, and a three-electrode system (Ag / AgCl as the reference electrode and Pt as the counter electrode). Figure 7 As shown in Figure A, the cathode photocurrent of PyTTA-HOF / MXene is significantly higher than that of PyTTA-HOF, indicating that this functionalization modification is beneficial to improving the electron-hole separation efficiency in photogenerated materials, thereby enhancing photoelectrochemical output. Furthermore, Figure 7 B in the diagram shows that the hydrogen peroxide electron acceptor can enhance the photoelectrochemical response of the PyTTA-HOF / MXene photoelectrode, and the response of the PyTTA-HOF / MXene photoelectrode to different concentrations of hydrogen peroxide (from 1.0 × 10⁻⁶) was investigated. -11 M to 1.0×10 -7 The photoelectric response of M) will be studied to further explore the feasibility of hydrogen peroxide as a photoelectric sensing signal molecule. For example... Figure 7As shown in Figure C, the photocurrent steadily increases with increasing hydrogen peroxide concentration, exhibiting a positive correlation between the logarithm of hydrogen peroxide concentration and the photocurrent. This provides an opportunity to establish a PEC sensing platform based on hydrogen peroxide-modulated PyTTA-HOF / MXene photoelectrode PEC response. Subsequent processing of the test data revealed ( Figure 7 (See the illustration in Figure C). There is a linear relationship between the logarithm of H₂O₂ concentration and photocurrent intensity, satisfying the equation I / μA = 4.86 + 0.210 log C / mol·L⁻¹. -1 (R) 2 =0.990). Figure 7 The D-prediction model suggests a plausible PEC sensing pathway: PyTTA-HOF / MXene can be excited to promote electron migration from the valence band (VB) to the conduction band (CB), generating photogenerated electron-hole pairs. These electrons can then be captured by dissolved oxygen to produce a photocurrent. PyTTA-HOF / MXene exhibits a stronger PEC response compared to PyTTA-HOF, attributed to MXene's excellent conductivity and charge transport capabilities. This mechanism effectively accelerates electron transport while suppressing electron-hole recombination. Furthermore, its layered structure provides a large surface area, which can disperse PyTTA-HOF and improve photon capture efficiency. The presence of hydrogen peroxide, acting as a highly efficient electron acceptor, rapidly removes photogenerated electrons to suppress charge recombination and accelerate charge separation, resulting in a significantly enhanced cathodic photocurrent.

[0055] (7) Quantitative detection of target DNA By integrating the hydrogen peroxide response capability of PyTTA-HOF / MXene, the DNA-specific recognition-mediated CRISPR / Cas12a system, and the signal transduction capability of MSP, a unified PEC sensing platform for the HPV-16 gene was designed and developed. Figure 8 As shown in Figure A, the photocurrent of the PEC sensing platform is significantly enhanced in the presence of the target, and the cathode signal intensity increases with the increase of the target HPV-16 level, confirming that the platform can convert target determination into a measurement based on GOx-driven hydrogen peroxide generation. Figure 8 (B in the middle). Figure 8 C in the figure illustrates the relationship between photocurrent intensity and target HPV-16 concentration, with results shown at 1.0 × 10⁻⁶. -14 Up to 1.0×10 -8 Within the M range, the photocurrent showed a good linear correlation with the logarithm of HPV-16 DNA concentration. The calculated limit of detection (LOD) was 4.8 × 10⁻⁶. -15M (Signal-to-noise ratio S / N=3). Compared with previously reported HPV-16 DNA detection methods (Table 2), this study demonstrated a wider linear range and a lower limit of detection (LOD). This superior performance stems from the synergistic effect of CRISPR / Cas12a system-driven homogeneous amplification and the excellent PEC performance of PyTTA-HOF / MXene.

[0056] Table 2 compares the proposed biosensing strategy with other reported HPV-16 detection methods.

[0057] [1] Zhang LL, Liu LB, Guo J, et al. Laser-induced WO3-decoratedporous graphene for portable and self-powered photoelectrochemicalaptasensing [J]. Microchimica Acta, 2025, 192: 682. [2] Zeng Y, Wang YH and Zhang Y Q. Review on Research Progress ofPhotoelectrochemical Biosensors [J]. 2025, 16(11): 1293. [3] Suo ZG, Yu TF, Xu YW, et al. Research progress of photoelectrochemical sensors in food detection [J]. 2025, 206: 116071. [4] Shi JJ, Chen ZC, Zhao CQ, et al. Photoelectrochemicalbiosensing platforms for tumor marker detection [J]. Coordination ChemistryReviews, 2022, 469: 214675. [5] Shan J J, Cheng Y, Zhang M, et al. Au-Ag@ZnS quantum dotsnanohybrid-enabled photoelectrochemical biosensing platform for highlysensitive detection of HPV 18 DNA [J]. Electrochimica Acta, 2025, 541:147368. [6] Tu Y P, Wang H H, Long D, et al. Highly sensitivephotoelectrochemical biosensor based on Au nanoparticles sensitized zincselenide quantum dots for DNA detection [J]. Sensors and Actuators B:Chemical, 2022, 357: 131255. [7] Cui Y B, Yan H, Sun Z, et al. A photoelectrochemical biosensorbased on ZnIn2S4@AuNPs coupled with circular bipedal DNA walker for signal-ondetection of circulating tumor DNA [J]. Biosensors and Bioelectronics, 2023,231: 115295. [8] Mo X X, Wang Y, Xiao Q Y , et al. Conjugated polymer sensitizedhyperbranched titanium dioxide based photoelectrochemical biosensor fordetecting AFP in serum [J]. Surfaces and Interfaces, 2021, 24: 101103. [9] Zhou M J, Cui Z Y , Liu Y, et al. Core-sheath-type TiO2@NH2-MIL-125(Ti) nanocone arrays for sensitive photoelectrochemical detection ofcarcinoembryonic antigen [J]. Chemical Engineering Journal, 2025, 519:165303.

[10] Xue Y L, Dong W S, Wang B, et al. A multifunctionalelectrochemiluminescence and photoelectrochemical biosensor based on aquantum dot ion-exchange reaction for two-channel detection of thrombin [J].Analyst, 2023,148: 4456-4462.

[11] Dong W S, Wang B, Xue Y L, et al. A multifunctionalphotoelectrochemical biosensing platform based on CdS QDs@TiO2-sensitizingNi-TBAPy-SC coupled with multiple amplification for detection of HBV andthrombin [J]. Sensors and Actuators B: Chemical, 2023, 393: 134293.

[12] Liu ZP, Li J, Gao LM, et al. Light-Addressable RegenerativePhotoelectrochemical Biosensor Array with Self-Calibration for High-Throughput and Accurate Detection of Circulating Tumor Cells [J]. AnalyticalChemistry, 2025, 97(23): 12364-12371. (8) Sensor stability and selectivity Specificity is an important indicator for evaluating the accuracy of biosensor detection results. Four analogues (HPV-18, HPV-31, HPV-51, and HPV-58) and a mixture of these four analogues with HPV-16 were selected to evaluate the selectivity of the constructed analytical method. Figure 8 The result shown in D represents the four interference signals (1.0 × 10⁻⁶). -10 M) is basically consistent with the blank signal, while for HPV-16 (1.0×10) -11 M) exhibited a significant photoelectric response. Subsequently, tests were conducted on samples containing four analogues (1.0 × 10⁻⁶). -10 M) and HPV-16 (1.0×10 -11 The photoelectric response of the mixture containing M was almost identical to that of the mixture containing only HPV-16, indicating that the presence of other interfering substances does not affect the sensor's detection of the target object. This demonstrates that the analytical method has strong anti-interference capabilities.

[0058] Furthermore, the stability of biosensor signals is another important indicator for evaluating sensor performance. Taking a photoelectrochemical sensor as an example, the stability of the signal at a depth of 1.0 × 10⁻⁶ is discussed. -13 The stability of the sensor was preliminarily evaluated through multiple consecutive measurements using M HPV-16. Experimental results showed ( Figure 8 In the photoelectric detection process, the photoelectric response corresponding to multiple consecutive photoswitches was recorded. The photoelectric response showed a relatively stable photocurrent intensity with an RSD of 3.94%. Figure 8 As shown in F, the storage stability of the prepared magnetic signal transduction probe was tested. After preparation, it was stored in a 4 ℃ refrigerator, and the stability was tested on days 1, 3, 5, 7, 11, 14, and 18 at a concentration of 1.0 × 10⁻⁶. -13The M HPV-16 was used for testing, and the results showed that the photocurrent intensity did not change significantly over at least two weeks, indicating that the probe used in this sensor can maintain good stability during storage. These results confirm the significant reproducibility and operability inherent in the proposed strategy.

[0059] (9) Actual sample testing The clinical application potential of the constructed PEC biosensor was evaluated using a spiking method, which involved adding target DNA (5.0 × 10⁻⁶) to normal human serum samples. -13 M, 5.0×10 -12 M and 5.0×10 -11 The recovery rate was tested using M), and the experimental results are shown in Table 3.

[0060] Table 3. Detection of HPV-16 in serum samples

[0061] As shown in Table 3, the recoveries ranged from 96% to 102%, with RSDs all below 5% (n = 3). These results... This indicates that the developed PEC biosensor has good potential for clinical application.

[0062] In summary, MXene-functionalized hydrogen-bonded organic frameworks (PyTTA-HOF / MXene) can be easily prepared using a solvent-induced self-assembly method. Systematic characterization confirmed that MXene functionalization significantly enhances the PEC performance of PyTTA-HOF. Gold nanoparticles loaded with GOx were designed as "signal-on" PEC tags that bind to streptavidin magnetic beads. By combining CRISPR / Cas12a system-driven homogeneous amplification via trans-cleavage with the excellent hydrogen peroxide PEC response of the PyTTA-HOF / MXene photoelectrode, this integrated scheme enables the detection of human papillomavirus nucleic acid (HPV-16) at 1.0 × 10⁻⁶. -14 Up to 1.0×10 - 8 Ultrasensitive quantitative detection within the M concentration range, with a detection limit (LOD) as low as 4.8 × 10⁻⁶. -15 M (Signal-to-noise ratio S / N=3). A solution-phase-based PEC biosensing platform without immobilization was successfully established. Benefiting from the combination of CRISPR / Cas12a system-mediated homogeneous biosensing and PyTTA-HOF / MXene photoluminescence within a single container, this strategy effectively bypasses cumbersome electrode modifications while significantly improving detection performance in a satisfactory manner.

[0063] More importantly, this sensing strategy exhibits remarkable flexibility and versatility—it is expected to be directly extended to other nucleic acid biomarkers by replacing the hairpin recognition domain. Therefore, this invention not only provides advanced photoactive materials and homogeneous sensing concepts for future photoelectrochemical biological detection, but also offers valuable insights for developing highly sensitive, user-friendly diagnostic tools.

[0064] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A homogeneous photoelectrochemical sensing platform, characterized in that, The product includes a working electrode modified with MXene-functionalized PyTTA-HOF material, a magnetic signal transduction probe, hairpin DNA, primer RNA, Cas12a enzyme, and buffer solution. The magnetic signal transduction probe comprises streptavidin magnetic beads and glucose oxidase-modified gold nanoparticles attached to their surfaces.

2. The homogeneous photoelectrochemical sensing platform according to claim 1, characterized in that, The streptavidin magnetic beads and glucose oxidase-modified gold nanoparticles are linked by a DNA linker. The DNA linker is modified with biotin at the 5' end and with thiol at the 3' end.

3. The homogeneous photoelectrochemical sensing platform according to claim 1, characterized in that, The Cas12a enzyme has trans-cleavage activity against DNA linkers; The buffer solution is a Tris-HCl buffer solution.

4. The homogeneous photoelectrochemical sensing platform according to claim 1, characterized in that, The method for preparing the working electrode modified with MXene-functionalized PyTTA-HOF material is as follows: add an aqueous solution of MXene-functionalized PyTTA-HOF material to the working electrode and dry it with infrared radiation. The preparation method of the MXene-functionalized PyTTA-HOF material is as follows: PyTTA monomer solution is added to MXene aqueous solution and stirred and mixed, then centrifuged, washed, and freeze-dried to obtain the material. The molar ratio of PyTTA to MXene is (3-4):1, preferably 3.6:

1.

5. The homogeneous photoelectrochemical sensing platform according to claim 1, characterized in that, The magnetic signal transduction probe is prepared by adding gold nanoparticle solution, DNA linker and streptavidin magnetic beads sequentially to glucose oxidase solution, shaking to mix, magnetically separating, washing and dispersing in buffer solution for later use.

6. The application of the homogeneous photoelectrochemical sensing platform according to any one of claims 1-5 in the detection of biological target molecules.

7. The application according to claim 6, characterized in that, The detection method for biological target molecules is as follows: The sample to be tested is mixed and incubated with hairpin DNA to prepare a mixed solution; Cas12a enzyme-primer RNA complex is added to the mixed solution, and after mixing and incubation, a magnetic signal transduction probe is added, and after mixing and incubation, magnetic separation is performed to obtain a supernatant containing glucose oxidase-modified gold nanoparticles; a buffer solution is added to the supernatant to prepare an electrolyte, and a three-electrode system with a working electrode modified with MXene-functionalized PyTTA-HOF material is used for photoelectric testing under ultraviolet irradiation. The content of biological target molecules in the sample to be tested is calculated according to the standard curve.

8. The application according to claim 7, characterized in that, The biological target molecule is human papillomavirus nucleic acid, which is one or more of HPV-16, HPV-18, HPV-31, HPV-51 and HPV-58, and the sample to be tested includes serum.

9. The application according to claim 7, characterized in that, Add the Cas12a enzyme-primer RNA complex and incubate for 20-100 min; add the magnetic signal transduction probe and incubate for 20-70 min. Preferably, the incubation time for adding the Cas12a enzyme-primer RNA complex is 60 min, and the incubation time for adding the magnetic signal transduction probe is 60 min.

10. The application according to claim 7, characterized in that, The wavelength of ultraviolet irradiation is 360-370 nm.

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